Various approaches have been reported to repair DNA using base excision enzymes. Unfortunately, these approaches in different ways cause further damage to the DNA. Conventional PCR techniques have been modified to improve amplification in some aspects. U.S. Pat. No. 5,035,996 describes a process for controlling contamination of nucleic acid amplification reactions that uses the modified nucleotide, dUTP, in the amplification reaction. This process uses uracil glycosylase to eliminate those PCR products containing uracil to prevent contaminating subsequent PCR reactions. U.S. patent publication no. 2004-0067559 A1 also relies on modified bases in primer DNA prior to amplification and uses, for example, dUTP for incorporation into the amplicon. The amplicon can then be fragmented by adding, for example, Uracil-DNA Glycosylase (UDG) and Endonucleaese (Endo) IV.
Hot start nucleic acid amplification has been used to lower mis-priming during PCR. One type of hot start amplification relies on the presence of a PCR primer with a blocked 3′ terminus to prevent extension by the polymerase present in the PCR reaction (see for example US 2003-0119150). The primer is unblocked by a thermostable 3′-5′ exonuclease that is active at >37° C. Therefore, the polymerase will only extend the PCR primers once the exonuclease unblocks the 3′ end at >37° C. Alternatively the Taq polymerase is blocked and then activated at amplification temperatures.
Barnes, W. M. Proc. Natl. Acad. Sci. USA 91:2216-2220 (1994) describes the use of vent polymerase and Taq polymerase as an improvement over the use of Taq polymerase only in amplification. Ghadessy et al. reported a mutant Taq polymerase that is not halted by damaged or abasic sites (Ghadessy et al. Nature Biotechnol. 22(6):755-9 (2004)).
It has been reported that conventional amplification techniques are compromised if the DNA is substantially damaged (DiBernardo et al. Nucl. Acids Res. 30:e16 (2002)). Degradation and/or fragmentation of DNA resulting from exposure to the environment and microorganisms which contain DNA endonucleases is a frequent problem in forensics, diagnostic tests and routine amplification and affects fidelity and yield of the amplification product. In addition, the problem of degraded DNA is also faced by researchers who are analyzing the DNA obtained from frozen, extinct or extremely rare organisms.
Fromenty, B., et al. Nucl. Acids Res. 28(11):e50 (2000) and International Publication No. WO/0151656 reported that Exonuclease (Exo) III improved yields of long PCR. Fromenty also reported decreased yields of amplicon for DNA<500 bp. One of the problems associated with the use of Exo III is that it degrades template and primers.
Di Benardo et al. Nucl. Acids Res. 30(4):e16 (2002) described the use of T4 DNA ligase (T4 ligase) and an E. coli polymerase to amplify short regions of single-stranded DNA between cross-linked regions of double-stranded DNA.
Another approach to amplification of damaged DNA has been described in U.S. Publication No. 2003-0077581. Degraded nucleic acid was hybridized to undegraded nucleic acid having a sequence homologous to the degraded nucleic acid. Regions of the degraded nucleic acid were then filled in with nucleotide precursors. The fragmented strands were then covalently linked using a polymerizing and/or ligating enzyme.
Preparations for improving amplification of damaged DNA can be obtained commercially from Sigma, St. Louis, Mo. and Qbiogene, now MP Biomedicals, Irvine, Calif. Although the compositions of these preparations are not provided, it is assumed that Exo III is contained in the preparation. The preparations are not recommended for DNA templates less than 500 base pairs in length.
Others report the use of a combination of E. coli DNA PolI and T4 ligase for pre-amplification repair (Pusch, et al., Nucl. Acids Res. 26:857 (1998)). However, according to Pusch et al. the preamplification product is purified before initiation of amplification.